Acute balenine supplementation in humans as a natural carnosinase-resistant alternative to carnosine

Balenine possesses some of carnosine’s and anserine’s functions, yet it appears more resistant to the hydrolysing CN1 enzyme. The aim of this study was to elucidate the stability of balenine in the systemic circulation and its bioavailability in humans following acute supplementation. Two experiments were conducted in which (in vitro) carnosine, anserine and balenine were added to plasma to compare degradation profiles and (in vivo) three increasing doses (1–4–10 mg/kg) of balenine were acutely administered to 6 human volunteers. Half-life of balenine (34.9 ± 14.6 min) was respectively 29.1 and 16.3 times longer than that of carnosine (1.20 ± 0.36 min, p = 0.0044) and anserine (2.14 ± 0.58 min, p = 0.0044). In vivo, 10 mg/kg of balenine elicited a peak plasma concentration (Cmax) of 28 µM, which was 4 and 18 times higher than with 4 (p = 0.0034) and 1 mg/kg (p = 0.0017), respectively. CN1 activity showed strong negative correlations with half-life (ρ = − 0.829; p = 0.0583), Cmax (r = − 0.938; p = 0.0372) and incremental area under the curve (r = − 0.825; p = 0.0433). Overall, balenine seems more resistant to CN1 hydrolysis resulting in better in vivo bioavailability, yet its degradation remains dependent on enzyme activity. Although a similar functionality as carnosine and anserine remains to be demonstrated, opportunities arise for balenine as nutraceutical or ergogenic aid.


Scientific Reports
| (2023) 13:6484 | https://doi.org/10.1038/s41598-023-33300-1 www.nature.com/scientificreports/ poor substrates for the CN1 enzyme [15][16][17] . In vivo human experiments show a rapid degradation of carnosine upon absorption. Ingestion of carnosine elicited a small increase in plasma concentration, but only in people with low CN1 enzyme activity and with a very high dose of 60 mg/kg bodyweight 18,19 . Ingesting anserine, on the other hand, elicited dose-dependent peak concentrations of 0.5-3.1 µM. Increasing plasma anserine appeared to pose less of a challenge. Nevertheless, up to a dose of 10 mg/kg, measurable plasma anserine was mainly observed in people with low CN1 enzyme activity 18,20 . It was shown that carnosine and anserine are absorbed intact into the bloodstream, but their short elimination half-life and low to undetectable concentrations in the human systemic circulation may be a major limitation for translating their therapeutic potential. Therefore, as balenine was shown in in vitro studies to have a low hydrolysis rate and its functionality partly resembles carnosine's and anserine's, it might be an even better nutraceutical. Hence, it is necessary to gain a better insight into its oral bioavailability and pharmacokinetic (PK) properties. The aim of this study was twofold: first, we tested balenine's degradation rate in vitro and hypothesized that it would be slow as it was hypothesized not to be a suitable substrate for CN1. Secondly, an in vivo PK study was conducted in which absorption, distribution, metabolism and excretion of balenine was studied. We optimized and validated an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify HCDs at low concentrations with high accuracy and precision. Population pharmacokinetic analysis was performed to gain insight in the best possible dosing regimens for future studies.

Results
In vitro experiment. Figure 1A shows the mean breakdown profile (relative to starting concentration) of the HCDs. Carnosine and anserine are rapidly hydrolysed upon addition to the heparin plasma and appear undetectable after 5 and 15 min, respectively. In contrast, balenine remains elevated above LOQ up to 5 h. The elimination constant, ke, presented a similar pattern (Fig. 1B). A Friedman test (p = 0.0001) showed a significantly lower ke for balenine (× 25.9; p = 0.0016) compared to carnosine. No significant differences were found between the ke of balenine and anserine (× 14.0; p = 0.250) or carnosine and anserine (× 1.8; p = 0.250). Half-life of carnosine (1.20 ± 0.36 min) was 29.1 times lower (p = 0.0044) than that of balenine (34.9 ± 14.6 min). Moreo- Figure 1. Mean breakdown profile of carnosine, anserine and balenine, relative to their starting concentration. The panel in the middle zooms in on the profile up to 10 min. Molecular structure of carnosine, anserine and balenine in which the methyl-group is indicated (A). The mean elimination rate constant (ke) of carnosine, anserine and balenine, and the factor difference between the dipeptides (B). Correlation between the half-life (t1/2) of the dipeptides in human plasma samples of different subjects and their respective CN1 enzyme activity (C). *p < 0.05. The correlation coefficient shows strong negative relations for the three HCDs between their half-life and CN1 activity, although only significant for balenine (carnosine: ρ = − 0.769; p = 0.0741; anserine: ρ = − 0.771; p = 0.0725; balenine: ρ = − 0.937; p = 0.0058) (Fig. 1C).
Since iAUC and Cmax in the 1 and 4 mg/kg dose correlated with body weight from which dose was determined, only the 10 mg/kg (fragmented dose) will be shown. For iAUC and Cmax, a significant negative correlation was found with CN1 enzyme activity (iAUC: r = − 0.938; p = 0.037, Cmax: r = − 0.825; p = 0.043; Fig. 2C) indicating that with higher activity, more balenine is hydrolysed. For time to peak concentration, no significant correlation was observed (r = − 0.559; p = 0.248).
Population pharmacokinetic modelling of balenine. A 1-compartment model with sequential zeroand first-order absorption and linear elimination was identified as the best model to describe plasma concentration-time profiles of balenine. The zero-order input lasted 1.13 h for the 1 and 4 mg/kg doses and was shorter for the 10 mg/kg dose. It was followed by a fast first-order absorption (ka = 3.48/h, fixed to its last estimated value, www.nature.com/scientificreports/ or an absorption half-life of 0.2 h). The volume of distribution (V) was estimated at 126 L, and the total clearance (CL) at 19.6 L/h (1 and 4 mg/kg), which is low. The PK parameters of balenine were dose-proportionally between the two lowest doses, but increased more than dose-proportional beyond 4 mg/kg. This translated in a lower volume of distribution and CL for the 10 mg/kg dose level. More than half of the balenine dose was eliminated unchanged through the kidney (56.4%). Inter-occasion variability, such as random variability in subjects' physiological parameters from one visit to the next or in the applied methods unrelated to the compound, was implemented on all parameters, and ranged from 0.11 to 0.38 (SD). Higher CN1 activity resulted in a significantly higher clearance, which is also reflected in the negative correlations with Cmax and iAUC, and a lower fraction excreted unchanged via the kidney. Pharmacokinetic parameters of the final model are summarized in Supplementary Table 1.

Discussion
In the present study, in vitro stability of balenine in human plasma and its in vivo bioavailability was investigated. The experiments were conducted with a particular interest in the relationship with CN1 activity and in comparison to the parent molecule carnosine and the alternatively methylated derivative anserine.
To measure balenine with high accuracy and precision, we optimized and validated an UHPLC-MS/MS method. In short, no carry-over was observed LOQ was 0.05 µM and linearity was confirmed up to 10 µM. Precision and accuracy were assessed within and between runs and passed the FDA requirements for analytical method development 21 . So, this method is validated to measure balenine accurately.
Previous in vitro research has shown that balenine is a poorer substrate for CN1 than carnosine and anserine 15,16 . This is corroborated by our in vitro study. Herein, the half-life of balenine was around 30 min while that of carnosine and anserine was only 1 and 2 min, respectively. Additionally, the reported rate of hydrolysis of balenine (relative to carnosine) was 3.81% 16 and 8% 15 . Our study confirms these results as the elimination rate constant of balenine was 3.86%. In addition to this confirmation of earlier findings, we hypothesized balenine to disappear more slowly from the systemic circulation with no relationship to CN1 activity, as compared to carnosine and anserine. However, elimination half-life and constant of balenine in vitro were negatively correlated to CN1 enzyme activity. In plasma with the highest CN1 activity, balenine had a half-life of 15 min for balenine while balenine in the plasma with the lowest enzyme activity had a threefold higher half-life. This indicates a strong effect of CN1 activity on elimination rate, which may explain high inter-individual differences in PK profile when balenine is acutely ingested. In addition to the in vitro findings, peak plasma concentration and iAUC following 10 mg/kg of balenine were also correlated with CN1 activity. This indicates that, although the CN1 enzyme has very low affinity towards balenine, its stability remains dependent on CN1 activity. These results counter our initial hypothesis.
In order to implement acute balenine ingestion as possible nutraceutical or performance enhancing supplement, its bioavailability after ingestion needs to be established. The main findings of the PK study are the high stability of balenine, as opposed to carnosine and anserine. When looking more closely to iAUC and peak plasma concentration of balenine, a non-linear higher increase in plasma balenine concentration is observed with higher doses. It is speculated that this results from a saturation of CN1, meaning that all binding sites for hydrolysis are occupied at a certain concentration. From then on, plasma balenine concentration increases exponentially 20 .
Since carnosine and anserine PK data is also available from previously published studies from our lab 20,22 , all data for oral ingestion of 1, 4, 10 or 20 mg/kg of carnosine, anserine or balenine in isolation, were integrated in Fig. 4. Carnosine was barely measurable, even after ingesting the highest dose of 20 mg/kg 22 . Cmax for anserine was 0.54 µM and 1.10 µM for the 4 and 10 mg/kg dose 20 but these concentrations were 18.5 and 25.9-fold higher when a same amount of balenine was ingested (Fig. 3A). Additionally, when comparing the plasma timecourse following ingestion of 10 mg/kg of either anserine or balenine, a very large difference in plasma iAUC is observed (Fig. 3B). For anserine, the iAUC was 66.7 µM.min while for balenine 8960 µM.min, which is a 136-fold www.nature.com/scientificreports/ difference. Overall, this shows that pure balenine is absorbed intactly with a much higher bioavailability than both carnosine and anserine, again indicating a much lower susceptibility to CN1 hydrolysis. When urinary dipeptide excretion is compared, the average excreted percentage of the dose is 6.3% for anserine but 27.4% for balenine (Fig. 4A). Interestingly, when comparing ingestion of 10 mg/kg of anserine and balenine, almost equal amounts of balenine are excreted in urine in the first hours following ingestion, while the plasma concentration of balenine, and thus the amount filtered, is a lot higher (Fig. 4B). A possible explanation relates to the affinity of HCDs to the human peptide transporter 2 (PEPT2) in the proximal kidney tubule. PEPT2 contributes to the reabsorption of filtered peptides 1 . Anserine is a high-affinity ligand for PEPT2 23 , which could result in high reabsorption from the filtrate in the kidney. The higher reabsorption in our study might indicate that balenine is an even better substrate for PEPT2. Indeed, some preliminary molecular dynamics experiments showed a possibility for improved tubular balenine reabsorption compared to anserine (Prof. Giulio Vistoli; personal communication). This higher reabsorption of balenine may also contribute to its increased bioavailability.
Carnosine was shown, mostly in rodents, to affect various pathologies such as multiple sclerosis 24 , Alzheimer's 25,26 , type II diabetes 27,28 , metabolic syndrome 29 and many other (reviewed by Artioli et al. 2 ). Although carnosine supplementation in humans occasionally appears beneficial [30][31][32] , translating its full potential remains a challenge, mainly because of the highly active CN1 enzyme in human circulation which is absent in rodents 14 . Balenine's high stability and bioavailability might be a way to overcome this translation difficulty. One prerequisite is that balenine's functionality should resemble that of carnosine. Although research into this topic is limited, it appears promising with evidence of an antioxidant capacity 7,11-13 , reaction with cytotoxic carbohydrate-derived aldehydes 33 and an effect on learning and memory in an Alzheimer's disease mouse model 34 . Future human studies should investigate whether balenine is indeed a better nutraceutical than carnosine and anserine, because of its superior stability and bioavailability. In addition to these possible health effects, balenine supplementation may also hold promise for ergogenic (sport performance) purposes. A negative correlation between CN1 activity and performance improvement indicates that bioavailability of the dipeptide is a prerequisite for improving performance 35 . Balenine could be a better alternative to carnosine and anserine supplementation in a sports setting as well.
Balenine is the first endogenous HCD that shows such good stability and bioavailability in humans. The fact that it is a naturally occurring compound in our diet makes the toxicology profile and therapeutic potential highly feasible. In addition, pharmacological variants of carnosine have been developed, such as Trolox-carnosine 36 , D-carnosine octylester 37 and carnosinol 29 . The latter is a reduced carnosine derivative that is resistant to CN1 and has shown promise to be used in the treatment of metabolic diseases of obesity and diabetes.
To conclude, this research corroborated previous findings stating that balenine is significantly more stable in vivo than anserine and carnosine. This was reflected in vivo in a higher peak plasma concentration and iAUC after supplementing with lower doses of balenine, compared to carnosine and anserine. Moreover, balenine stability appears to be dependent on serum CN1 enzyme activity. Since balenine has a somewhat similar functionality as carnosine and anserine, this might generate much better opportunities for developing balenine as nutraceutical or ergogenic aid, when translating animal research to humans.

Methods
This paper includes an in vitro and in vivo pharmacokinetics (PK) experiment to determine absorption, degradation and elimination of balenine, carnosine and anserine in plasma with varying CN1 enzyme activity. All subjects gave their written informed consent and the study was approved by the Local Ethics Committee of the Ghent University Hospital (BC07532). It was performed in accordance with the standards of ethics outlined in the Declaration of Helsinki.
Pure, chemically synthesized carnosine and anserine were kindly provided by Flamma S.p.a. (Chignolo d'Isola, Bergamo, Italy) and the balenine powder by NNB Nutrition (Frisco, Texas, USA). with a wide range in CN1 enzyme activity (0.32-2.92 µmol/mL/h with a mean value of 1.62 µmol/mL/h). Subjects were asked to refrain from alcohol, meat and fish the day before the sampling. In separate tubes, a final concentration of 20 µM of carnosine, anserine and balenine were added to the plasma, which was kept at 37 °C. On different time points (Table 1), plasma was deproteinized with 1:11 sulfosalicylic acid (SSA, 35% solution) to inhibit further hydrolysis. Samples were centrifuged for 5 min at 16,000 g and supernatant was stored at − 20 °C until further UHPLC-MS/MS analysis.
In vivo PK experiment. Since this study is the first to supplement pure, chemically synthesized balenine to humans, a thorough safety evaluation was conducted by Vedic Lifesciences (Mumbai, India) (190,911/WGI/ PC). Seven female Sprague Dawley rats (8-12 weeks old) were acutely supplemented with increasing doses of balenine up to 2000 mg/kg. Following treatment, a 2 week observation period with twice daily examination for morbidity and mortality was performed. No pathological changes were observed in any animal. It was concluded that a dose of 2000 mg/kg balenine was safe to use in the following experiments. Six subjects (3 males and 3 females) participated in this PK study in which three increasing doses were tested. For the third dose, one female was replaced, due to pregnancy, by another woman with similar CN1 activity. Their mean (n = 7) weight, height and age were 66.4 ± 10.1 kg, 171 ± 12 cm and 25.4 ± 1.7 years. CN1 activity ranged between 0.63 and 3.74 µmol/mL/h with a mean of 2.43 µmol/mL/h. Since this was the first time pure balenine was ingested by human volunteers, no dose randomization, but a gradual increase in dose was opted for. On the first test day, subjects received 1 mg/kg bodyweight of balenine and 4 mg/kg the following test day. Test days were separated by at least two weeks to ensure complete return to baseline balenine concentration. After analysing these samples, a third test day was added in which 10 mg/kg in a fragmented dose was provided to exclude the effect of bodyweight on balenine concentration in plasma (Stautemas et al. 2019). This means that half of the dose was administered as 5 mg/kg (weight-relative part) and the other half was an absolute dose based on the mean weight of the subjects of the third test day (341 mg; fixed part).
The day before a test day, subjects were asked to refrain from meat and fish. Upon arrival, a catheter was inserted in an antecubital vein and a fasted ethylenediaminetetraacetic acid (EDTA, 4 mL) and serum sample (3.5 mL, to determine CN1 activity) were collected. Then they were asked to empty their bladder, after which the supplement, being 1, 4 or 10 mg/kg balenine in gelatine capsules was ingested. The following 2 h, every 30 min an EDTA blood sample was taken. Then after 3, 4, 5, 8 and 24 h, another sample was withdrawn. Urine was collected after 150 min, 5, 8 and 24 h. The subjects arrived in the lab and remained fasted until 2 h after ingesting the supplement. Subjects remained in the lab until the post 8 h blood sample, and returned the following morning for the post 24 h sample. Throughout the test day, food intake was standardized (lacto-ovo-vegetarian diet) ensuring minimal HCD intake for 24 h following supplement. Figure 5 gives a schematic overview of a test day.

Time points (min
In the in vitro study, supernatant was diluted 40 times before injection. In the PK study, plasma samples from the 10 mg/kg condition and baseline urine samples were diluted 100 times. All other urine samples were diluted 1000 times. Dilutions were conducted with acetonitrile containing 1% formic acid. Pooled heparin (in vitro study) and EDTA (in vivo study) plasma, and urine were used to prepare calibration curve and QC samples (identical extraction protocol as samples). For quantification, the following concentration ranges were used: in vitro study and 1 and 4 mg/kg condition: 0-25 µM with QC at 0.05, 0.5 and 5 µM; 10 mg/kg condition: 0-100 µM with a fourth QC of 50 µM; urine: 0-60 µM with QC at 0.05, 0.5, 5 and 50 µM.  Table 3. Instrument parameters were optimised by direct infusion of working solution of 100 ng/mL of carnosine, anserine, balenine and IS at a flow rate of 10 µL/min and in combination with the mobile phase (15% A, 80% B, 5% C) at a flow rate of 300 µL/min. The detector operated in multiple reacting monitoring mode, scanning the two most intense transitions of the HCDs. Masslynx software 4.2 was used for instrument control and data extraction. Hardware and software were purchased from Waters (Milford, MA, USA).

UHPLC-MS/MS method validation for the quantification of balenine.
This method was validated on three separate days according to the US FDA guidelines for bio-analytical method validation 21 . The following parameters were considered: selectivity, linearity, within-and between-run precision and accuracy, and carryover effect.
Plasma samples, withdrawn after two days of lacto-ovo-vegetarian diet, showed a small endogenous peak at the retention time of balenine. Nevertheless, the method is considered selective since the chromatographic peak area of this sample is on average 5.79% of the area of balenine at 0.05 µM (limit of quantification; LOQ). This is lower than 20% at LOQ level, as indicated by the FDA guidelines. Linearity of the calibration curve was good from 0.05 µM to 10 µM. The equation y = ax + b, with a the slope and b the intercept (mean ± SD), was y = 0.00358 (± 0.00038)x + 0.00577 (± 0.00818) with r = 0.9974 (± 0.0011) and a goodness of fit of 6.49 (± 0.98)%. No carryover was observed following the highest QC and calibration samples. Within and between-run precision and accuracy were evaluated by analysing three concentrations (0.05-0.5-5 µM) six and two times, respectively, on three separate occasions. Results are summarized in Table 4.
Serum carnosinase (CN1) activity. CN1 activity was quantified by fluorometric determination of histidine following carnosine addition (based on Teufel et al., 2003). The reaction was initiated by adding carnosine to the serum (final concentration of 3.33 mM carnosine) and stopped after 10 min incubation at 37 °C by adding trichloroacetic acid (final concentration of 150 mM TCA). For controls, trichloroacetic acid was added before carnosine. After centrifugation (15 min, 16000 g), 10 µl supernatant was added to 190 µl of an OPA-NaOH mixture (4000 µl incomplete ophtaldehyde, with 0.2% 2-mercaptoethanol, and 15 ml of 4 M sodium hydroxide solu- Calculations and statistical analysis. HCD concentration in the in vitro experiment was calculated relative to the starting concentration. Elimination constant (ke) and elimination half-life were calculated based on a monophasic decay profile. Incremental area under the plasma concentration time curve (iAUC) was calculated using the trapezoidal method in the PK study. A normality check was performed using Shapiro-Wilk's test. In case of normally distributed data, a mixed-effects model and Pearson correlation was used. Skewed data were analysed with Friedman tests and Spearman rank correlations. Values are presented as mean/median ± standard deviation/inter quartile range and 95% confidence intervals (95% CI). Significance is assumed at p ≤ 0.05. Graphpad Prism (version 9; GraphPad Software, San Diego, California) was used for statistical analyses. Plasma and urine balenine concentrations (in vivo) were analysed using a nonlinear mixed-effects modelling approach as implemented in the Monolix software (Version 2021R1; Lixoft, Antony, France). Plasma concentrations were described using a 1-compartmental model, with sequential zero-and first-order absorption and linear elimination. Cumulative urine concentrations were fitted simultaneously with plasma concentrations. Inter-occasion variability was also implemented and assumed to follow a lognormal distribution. Residual variability was described using a combined error model, consisting of an additive and a proportional component. The following covariates were tested: sex, age, weight, CN1 activity and dose. Model evaluation was done based on standard goodness-of-fit metrics (objective function value, standard errors) and plots.

Data availability
Raw data were generated at the Sports Science Laboratory, Jacques Rogge, Watersportlaan 2, 9000 Ghent, Belgium. Derived data supporting the findings of this study are available from the corresponding author WD on request.  Table 4. Results of the within and between-run precision and accuracy evaluation for the analysis of balenine in plasma. a within-run analysis (n = 6; on 3 separate days); b between-run analysis (n = 2; on 3 separate days); SD Standard deviation, RSD Relative standard deviation.

Funding
Sarah de Jager is a recipient of a PhD fellowship by Research Foundation Flanders (FWO).